There are two very widely used and effective drugs for the treatment of Trypanosoma cruzi infection - benznidazole and nifurtimox. Although the conditions under which these 2 drugs actually achieve parasitological cure in hosts is uncertain - due to the difficulty of detection T. cruzi, particularly in chronically infected host - the fact tat these compounds can achieve cure is not debated. The purpose of this application is to determine why benznidazole (BZ) fails to achieve full infection control in some cases and based on these results, move toward the development of better protocols of treatment that will yield a much higher success rate. BZ is a highly effective drug, and rapidly kills the vast majority of T. cruzi in a host with a single dose of treatment. Nevertheless, consistent cure of the infection requires 40 days of treatment with BZ-sensitive strains and fails to cure infection with BZ-resistant strains. These data suggest that at any one time during T. cruzi infection, there are parasites within host cells that are, for whatever reason, inaccessible to drug. The primary hypothesis to be tested in this application is that both the requirement of a long course of treatment for cure of T. cruzi with most strains and the resistance of some strains to BZ-induced cure, is due to the propensity of T. cruzi to invade host cells that are inaccessible to drug. The alternative hypothesis, that a subset of the population of T. cruzi in infected hosts is dormant and as a result insensitive to drug, will also be explored. Depending on the mechanism of in vivo resistance to BZ treatment, this project will also explore protocols that will improve treatment success rate for both BZ-sensitive and BZ-resistant T. cruzi strains while also minimizing dosage of drug delivered and thus the potential for adverse events. The most rapid path to better treatments for Chagas disease is to optimize the delivery of known, highly effective drugs such as BZ. This project will accomplish that goal while also generating basic information on the biology of T. cruzi infection that will help guide the development of even better treatment protocols and more highly effective drugs.